Expression of recombinant mature human tyrosinase from Escherichia coli and exhibition of its activity without phosphorylation or glycosylation.

A cDNA encoding mature human tyrosinase was cloned into pET-23a(+) and transformed into E. coli BL21(DE3). Three major recombinant proteins, mature human tyrosinase (RHT₂₀₋₅₃₁), N-terminal truncated human tyrosinase (RHT₁₆₈₋₅₃₁), and β-lactamase, were overexpressed as inclusion bodies in E. coli after 12 h of induction with 1.0 mM isopropyl-β-D-thiogalactopyranoside at 37 °C. After sonication and centrifugation, the inclusion body was harvested, solubilized, dialyzed, and refolded into the active form with monophenolase and diphenolase activities. It was purified to homogeneity by DEAE-Sepharose FF and Sephadex G-75. The molecular mass and N-terminal sequence were 57.0 kDa and GHFPRAC, respectively, and corresponded to those of mature human tyrosinase. The RHT was active in a broad range of temperature and pH, and with optimum activity at 70 °C and pH 8.5.